Dr. Kays invokes a mitochondrial DNA (mtDNA) capture hypothesis to explain potential discordances between mtDNA haplotype and complete genome sequence data. However, the presence of a divergent mtDNA haplotype is not evidence enough to support the mtDNA capture hypothesis or the existence of a separate species. Caution is warranted when interpreting results from mtDNA sequences as they derive from a single non-recombining locus that only represents matrilineal dynamics and may be affected by natural selection (e.g. 1). It is well known that gene genealogies are highly variable due to the randomness inherent in the coalescent process (e.g. 2-4). In other words, a single demographic model can and will generate very different genealogies. Divergence and recent branches within a genealogy are not surprising and are even expected. Further, this dichotomy becomes more pronounced with large ancestral population sizes (N), as the variance in the time to the most recent common ancestor is of the order N2 (3-5).

Gray wolves and coyotes are known to have had a large ancestral population size (e.g. effective size 30,000-45,000 for wolves, 6). Therefore, coalescent processes can generate divergent lineages as well as clusters of closely related mtDNA haplotypes. A divergent lineage, if examined in isolation, could be misinterpreted as support for a distinct species if mtDNA sampling was geographically restricted. The divergent and controversial “Algonquin” or eastern wolf (Canis...

Dr. Kays invokes a mitochondrial DNA (mtDNA) capture hypothesis to explain potential discordances between mtDNA haplotype and complete genome sequence data. However, the presence of a divergent mtDNA haplotype is not evidence enough to support the mtDNA capture hypothesis or the existence of a separate species. Caution is warranted when interpreting results from mtDNA sequences as they derive from a single non-recombining locus that only represents matrilineal dynamics and may be affected by natural selection (e.g. 1). It is well known that gene genealogies are highly variable due to the randomness inherent in the coalescent process (e.g. 2-4). In other words, a single demographic model can and will generate very different genealogies. Divergence and recent branches within a genealogy are not surprising and are even expected. Further, this dichotomy becomes more pronounced with large ancestral population sizes (N), as the variance in the time to the most recent common ancestor is of the order N2 (3-5).

Gray wolves and coyotes are known to have had a large ancestral population size (e.g. effective size 30,000-45,000 for wolves, 6). Therefore, coalescent processes can generate divergent lineages as well as clusters of closely related mtDNA haplotypes. A divergent lineage, if examined in isolation, could be misinterpreted as support for a distinct species if mtDNA sampling was geographically restricted. The divergent and controversial “Algonquin” or eastern wolf (Canis lyacaon) carries a distinct mitochondrial haplotype, but is otherwise admixed with coyotes and gray wolves, and shares their mitochondrial haplotypes (7-9). Similarly, the red wolf lineage (Canis rufus) segregates coyote and wolf haplotypes but, under various sampling schemes, haplotypes are clustered within the clade of coyotes (10,11). As the analyses to date of the divergent eastern wolf haplotype only include individuals sampled from a relatively restricted geographic range, a more extensive geographic sample of haplotypes is necessary to test if a simple neutral null model explains the divergence of the Algonquin haplotype. In fact, the divergent Algonquin clade appears to represent one of several divergent clades in the coyote tree (8). For example, in one extensive study of coyotes that intentionally did not include canids from admixture zones, over 200 coyote haplotypes in 837 coyote samples were found representing several divergent mtDNA haplogroups (12).

Complex demographic scenarios, such as mtDNA capture, cannot be supported by sparse mtDNA data and are better tested by genome-wide data (e.g. 1). For example, if the coalescent-based question of our own human evolutionary origins is investigated with mtDNA data alone, there is evidence for explosive population growth since the late Pleistocene (e.g. 13) and no sign of Neanderthal admixture (e.g. 4, 14-17). It is only when nuclear data from many evolutionarily independent genomic regions are explored that explosive population growth is found to be much more recent than previously thought (e.g. 18, 19), with complex and variable patterns of Neanderthal admixture across different human populations (e.g. 20-23).

We believe that the invocation of an elaborate model of mitochondrial capture, which requires introgression to have incurred independently in two geographic regions that are now coincidently admixture zones, without first rejecting simpler null demographic models is both inappropriate and less parsimonious. Though mitochondrial capture is an intriguing hypothesis, this notion must be tested with coalescent simulations under various demographic models. Otherwise, a single genealogical tree is subject to over interpretation. Complete nuclear genome sequences provide a richer source of gene genealogies to test past admixture, and our analysis of whole genome sequence data finds no evidence of ancient wolf or coyote introgression.

A hypothesis for the disagreement between nuclear and mitochondrial DNA of North American Canis

RolandKays, Scientist, North Carolina Museum of Natural Sciences and North Carolina State University

(12 August 2016)

The whole-genome analysis of VonHoldt et al. (1) provides the most complete picture to date of North American Canis autosomes, finding no history for long independent, species level evolutionary histories for the red wolf (Canis rufus) or eastern wolf (C. lycaon), suggesting instead that both have a modern origin through hybridization between wolves (C. lupus) and coyotes (C. latrans). This compelling nuclear DNA evidence leaves one mystery yet unsolved - why do many eastern canids have a unique mitochondrial haplotype that does not match extant coyotes or wolves?
This 'lycaon-type' haplotype was the most compelling evidence used to argue that eastern wolves should be recognized as a unique species (2) because it is similar to coyotes, yet different enough (3%) to suggest ~500K years of divergence between the two. Thus, the eastern wolf was proposed as a sister species to the coyote, evolving into a larger, deer-eating predator in eastern forests. Samples from historic and fossil eastern wolves have likewise returned mtDNA haplotypes similar to this lycaon-type (3) or to coyotes (4, 5), further supporting the coyote sister species hypothesis. Red wolves also have coyote-like mtDNA (6). Thus while the nuclear genome results of VonHoldt et al. (1) support a two species model for North American Canids (C. lupus & latrans), the mtDNA patterns support a three or four species model (adding C. lycaon and/or rufus).

The whole-genome analysis of VonHoldt et al. (1) provides the most complete picture to date of North American Canis autosomes, finding no history for long independent, species level evolutionary histories for the red wolf (Canis rufus) or eastern wolf (C. lycaon), suggesting instead that both have a modern origin through hybridization between wolves (C. lupus) and coyotes (C. latrans). This compelling nuclear DNA evidence leaves one mystery yet unsolved - why do many eastern canids have a unique mitochondrial haplotype that does not match extant coyotes or wolves?
This 'lycaon-type' haplotype was the most compelling evidence used to argue that eastern wolves should be recognized as a unique species (2) because it is similar to coyotes, yet different enough (3%) to suggest ~500K years of divergence between the two. Thus, the eastern wolf was proposed as a sister species to the coyote, evolving into a larger, deer-eating predator in eastern forests. Samples from historic and fossil eastern wolves have likewise returned mtDNA haplotypes similar to this lycaon-type (3) or to coyotes (4, 5), further supporting the coyote sister species hypothesis. Red wolves also have coyote-like mtDNA (6). Thus while the nuclear genome results of VonHoldt et al. (1) support a two species model for North American Canids (C. lupus & latrans), the mtDNA patterns support a three or four species model (adding C. lycaon and/or rufus).

I suggest that the discordance between these two lines of evidence can be resolved through a "mitochondrial capture" hypothesis. This is an evolutionary scenario in which mtDNA is exchanged between two different species through an ancient hybridization event, followed by extensive backcrossing with one of the parental species (figure 1). The nuclear signature of the old hybridization event is thus swamped out by successive chromosomal crossover events in each back-crossed generation, and potentially, also removed by natural selection. Meanwhile, the non-recombinant, neutral, genetic mtDNA material originally exchanged between species in the hybridization event could persist, slowly evolving into its own unique form through genetic drift. This hypothesis has not been formally evaluated for North American canids but has been suggested to explain discordant patterns of nuclear and mtDNA in a variety of mammals including chipmunks (7), primates (8, 9), and bats (10).

Under the mitochondrial capture hypothesis, the lycaon-type mtDNA haplotype would have been 'captured' by wolf populations through an ancient hybridization event with the ancestors of modern coyotes. Over the intervening tens of thousands of years the coyote-like mtDNA haplotype within wolf populations would have slowly diverged through neutral evolution, as would the haplotypes within the ancient coyotes, leading to the 3% divergence we see today between the lycaon-type and modern coyote(6). The result is a modern population of eastern wolves that contains a mixture of mtDNA haplotypes showing ancestry from C. lupus, C. latrans, as well as the lycaon-type (11–13).

Modern genomics has shown that the speciation process is a lot messier than previously thought, with ancient hybridization often detected between closely related lineages, including our own genus (14). Indeed, there are numerous other lines of evidence supporting ancient hybridization between North American canids including the introgression of dog coat color genes into wolves and coyotes (15) and the massive mismatch in dating the divergence between coyotes and wolves based on nuclear and mtDNA (1). Considering the genetic patterns of North American Canis to be the result of an ancient mitochondrial capture event helps resolve long standing confusion over conflicting evidence, and adds support to the two species model suggested by VonHoldt et al (1).